I would like to prepare by my self a gradient gel to separate proteines. I did some research but I don't have the right equipment to do it, such as cylindres or the chamber system adequate for this procedure. Do you know any other practice method?
Actually I cannot imagine any alternative to both the cylinders and a casting chamber with downward hose outlet to reproducibly cast gradient gels without the doubt of preparation being done correctly.
If polystirene ready-made cylinders cannot be purchased, maybe a in-house system done with plastic cylinders interconnected with tubing may work. Also a casting chamber with gel inlet from the bottom is needed and may be worked out even if with a bit more cumbersome process.
Hello Blerta Stringa I also don't have proper equipment to prepare gradient gel. Please share your experience and procedure of making gradient gel. I want to prepare 4-12% gradient gel. Thank you..
Hi @Iqra .N , I also needed the same thing and there is a video on youtube about how to pour a gradient gel by just using a serological pipet. It might wont be great in your first try but you'll get use to it. It really works, this Lorberbaum guy is a genius.
https://youtu.be/zu5a-kpMK8k
Hi Ezgi Altun thank you for sharing link. Please share your recipe of gel. Do you use same sds page running , transfer buffer and voltage conditions for gradient gel?
Ezgi Altun , could you please share your protocol as Iqra Nadeem asked? I am trying to do the same.
Thank you very much for your kindness!
I'm sorry @Iqra .N , I forgot I didnt answer you. @Monara Kaélle Angelim I prepare 8-16% gradient gel and it is useful to nicely separate proteins between 200 kDa-10 kDa. But you can calculate the 30% polyacrylamide(or whatever percent polyacrylamide you use) amount and arrange the percentage of your gels as you wish. the more dense the gel, the more polyacrylamide you need to use and less ddH2O(because it should be 5 ml in total-be careful with the total volume when calculating the percentage) Here is the protocol:
8-16% Gradient Separating Gel and 5% Stacking Gel recipes:
8% separating gel (5 ml for 2 gels):
ddH2O: 3 ml
3M Tris-HCl, pH 8.9: 625 µl
20% SDS: 25 µl
30% Polyacrylamide: 1.335 ml
20% APS: 37.5 µl
TEMED: 2.5 µl
16% separating gel (5 ml for 2 gels):
ddH2O: 1.665 ml
3M Tris-HCl, pH 8.9: 625 µl
20% SDS: 25 µl
30% Polyacrylamide: 2.67 ml
20% APS: 37.5 µl
TEMED: 2.5 µl
First, 2.5 ml of 8% solution was pipetted into the serological pipette, then 2.5ml of 16% solution was added to the same pipette. At last, one small air bubble was introduced to the end of the pipette and travelled along it until the end of the pipette. The gradient mix was added very slowly into the gel assemble. (Like in the video)
*right after pouring the gel, add butanol to prevent bubbles in the gel. After it polymerizes, clean the butanol and wash with ddwater and add the stacking gel.
5% stacking gel (5 ml for 2 gels):
ddH2O: 3.85 ml
1M Tris-HCl, pH 6.8: 250 µl
20% SDS: 10 µl
30% Polyacrylamide: 850 µl
20% APS: 37.5 µl
TEMED: 2.5 µl
*put the 1mm 10 well comb immediately after pouring the stacking gel. And dont wait too long after it polymerizes(dont let the gel dry out). Put the gel in the running buffer immediately.
6X Reducing loading dye:
375 Mm Tris-HCl
6% SDS
50% Glycerol
0.03% Bromophenol Blue
2-mercaptoethanol
I put 4 µl dye in 14 µl sample for each well. So I load 18 µl to each well in total, if I prepared 1mm-10 well gel. Reducing samples should be boiled at 95 degree for 5 minutes prior to loading. Run at 50 mA for approcimately 40 minutes(for 1 gel). I'd recommend to use prestained marker in order to follow the bands and stop the run before smallest bands escape the gel.
*After run, gel should be stained with a solution of 2.5% Coomassie brilliant blue G-250, 45%metoh 10%acetic acid for 1h, and destained in 45%metoh 10%acetic acid overnight. If you use R-250, be prepared to destain it for 2 days or so.
One simiple method is to prepare 2 solutions one high (12%) and one low (4%)of acrilamide. Then using a 5-10 ml seriological pipette such up half what you need for your gel from the low then half from the high. Then with both solutions in the pipette suck an air bubble into the end and allow it to travel up the pippet. This mixes your gradient .. now pipette slowly into your glass plates...
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